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Methods in Pharmacology and Toxicology Gary W. Caldwell Zhengyin Yan Editors Optimization in Drug Discovery In Vitro Methods Second Edition M P ETHODS IN HARMACOLOGY T AND OXICOLOGY Series Editor Y. James Kang University of Louisville School of Medicine Prospect, Kentucky, USA For further volumes: http://www.springer.com/series/7653 Optimization in Drug Discovery In Vitro Methods Second Edition Edited by Gary W. Caldwell and Zhengyin Yan CREATe Analytical Sciences, Janssen Research & Development, LLC, Spring House, PA, USA Editors Gary W. Caldwell Zhengyin Yan CREATe Analytical Sciences CREATe Analytical Sciences Janssen Research & Development, LLC Janssen Research & Development, LLC Spring House, PA, USA Spring House, PA, USA ISSN 1557-2153 ISSN 1940-6053 (electronic) ISBN 978-1-62703-741-9 ISBN 978-1-62703-742-6 (eBook) DOI 10.1007/978-1-62703-742-6 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2013953234 © Springer Science+Business Media New York 2 014 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. 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While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com) Pref ace The discovery and commercialization of new drugs for humans is extremely complex. Typically, pharmaceutical companies have approached this pharmacology challenge by dividing the problem into three stages. These stages in a pharmaceutical drug development process are the discovery of drug candidates interacting at a particular therapeutic target using whole-animal models, the development of these drug candidates into new chemical entities (NCEs) using human subjects, and the commercialization of NCEs into medicines. The process requires an enormous fi nancial investment since a decade or longer is typically required to transform a drug candidate into a medicine. In addition and probably most important is that the process requires a large interdisciplinary team of scientists and support staff working closely together with a focused management team to be successful. Whole-animal i n vivo pharmacology models, which are required by drug regulatory agencies, are the gold standard for biopharmaceutic, pharmacokinetic, toxicokinetic, and pharmacodynamic late stage drug candidate predictions; however, for many biological pathways and mechanisms, they do not provide a good extrapolation to humans. To address this issue, pharmaceutical scientists have used an i n vitro and i n situ surrogate assay reductionisms approach to understand biopharmaceutic, pharmacokinetic, toxicokinetic, and pharmacodynamic properties and thus to select drug candidates that have a high probability of becoming an NCE and eventually a medicine (Fig. 1 ). These surrogate assays provide more representative methods to rule out adverse effects early in the screening process for new drug candidates and to provide a knowledge platform for the correlation of whole-a nimal i n vivo pharmacology results to humans. In this strategy, surrogate assays have been developed to understand the biopharmaceutics of drug candidates including the solid-state characteristics of the drug in physiological fl uids; that is, dissolution rates, dissociation constants, ionization potential, lipophilic partition coeffi - cients, hydrophobicity, stability, solubility, formulation, and permeability. The pharmacokinetics and toxicokinetics of drug candidates have been addressed by examining individual physiological processes such as absorption (i.e., passive, active, effl ux transport of drugs), distribution (i.e., tissue, protein, and cell drug binding), metabolism (i.e., cytochrome P450 (CYP) and UDP-g lucuronosyltransferases (UGT)), and drug excretion mechanisms (i.e., metabolism, renal and bile). The overall pharmacodynamic predictions of the drug candidates have been rationalized by receptor binding and functional assays and safety assessment assays including CYP inhibition and induction, drug–drug interactions via assessment of reactive metabolites, hERG (the human Ether-à-go-go-Related Gene), DNA damage, genotoxicity, and mutagenicity assays. Thus, based on this reductionism approach, the pharmacology of a drug can be under- stood and examined by studying its parts, such as biopharmaceutics, pharmacokinetics, toxicokinetics, and pharmacodynamics. As previously mentioned, each sub-part contained in Fig. 1 can be further subdivided into i n vitro and in situ surrogate assays. Due to the v vi Preface Fig. 1 Pharmacology of a drug large number of drug candidates that need to be tested, drug discovery and development groups in the pharmaceutical industry have adopted an assay tiered approach toward select- ing potential new drug candidates with superior drug properties from large compound collections; that is, funneling thousands of compounds through a series of high-throughput capacity assays to lower capacity assays, which reveal more and more detailed information on a particular sub-part of the reductionism scheme. Using this approach, “drug-like” characteristics in addition to effi cacy properties and good safety profi les are achieved. With this process in mind, the book, Optimization in Drug Discovery: In Vitro Methods , fi rst published in 2004, presented a compilation of detailed experimental protocols necessary for setting up a variety of i n vitro and in situ assays important in the selection of drug candidates based on the reductionism scheme outlined in Fig. 1 . Each chapter contained Introductions, Materials, Methods, Notes, and References sections providing scientists with important background information on the assay, a list of all the equipment and reagents necessary to carry out the assay, a step-by-step protocol, information on dealing with com- mon and unexpected experimental problems in the assay and fi nally, a listing of important supplementary readings. We now have compiled a second edition following the same format as the fi rst edition, which contains updated variations on previously reported assays and many new protocols. A total of 34 chapters have been contributed by experts covering a wide spectrum of subjects Preface vii including formulation, plasma binding, absorption and permeability, cytochrome P450 (CYP) and UDP-glucuronosyltransferases (UGT) metabolism, CYP inhibition and induction, drug transporters, drug–drug interactions via assessment of reactive metabolites, genotoxicity, and chemical and photo-mutagenicity assays. Since biopharmaceutic, pharmacokinetic, toxicokinetic, and pharmacodynamic are all interrelated, it has been long recognized that a series of i n vitro and i n situ assays are required to understand how to develop “drug-like” characteristics in new drug candidates. For example, biopharmaceutic parameters infl uence the transfer of a drug across cell membranes, and thus affect absorption and distribution of the drug, which in turn affects pharmacokinetic properties, which in turn affects pharmacodynamic properties and so on. Chapters 1 – 3 of the new second edition provide experimental methods for preparing an optimal drug formulation, measuring protein binding and red blood cell binding. When combined with measuring p K , solubility, lipophilicity, and plasma protein binding tech- a niques from Chaps. 1 , 8 , and 9 in the fi rst edition, the most fundamental physicochemical properties of a drug candidate can be determined. Having a good absorption profi le for new drug candidates is another important require- ment for a drug to be effective. Drug absorption is primarily governed by solubility properties of the solid neat drug, permeability, and infl ux and effl ux transport mechanisms. Chapters 4 and 5 are included in the second edition to address different issues on this aspect using a 5-day cultured Caco-2 cell model and an in situ single pass perfused rat intestinal model. Combining these assays with absorption models described in Chaps. 2 – 5 in the fi rst edition, the most commonly used assays to investigate drug absorption mechanisms are available to research scientists. Optimal metabolic stability of new drug candidates is one property that is necessary for a drug to have a long systemic half-life in the body and thus, lasting pharmacological effects on the action site. There are many different i n vitro metabolic stability assays that can be used to understand the metabolism fate of new drug candidates, identify potent metabolites with better “drug-like” properties, and for using metabolic stability information to guide new synthesis and generate more stable drug candidates. In C haps. 6 and 7 in the second edition, the assessment of CYPs and UGTs metabolism is determined from incubations with either hepatocytes or microsomes. C hapters 8 and 9 in the second edition outline assays to determine the CYP and UGT phenotyping. When these assays are combined with metabolic stability assays from Chaps. 1 0 – 1 2 in the fi rst edition, an arsenal of assays are available with clear advantages and objectives to address most metabolic stability questions. Drug–drug interactions (DDIs) are defi ned when one drug alters the pharmacokinetics or pharmacodynamics of another drug. Since biopharmaceutic, pharmacokinetic, toxicokinetic, and pharmacodynamic are all interrelated, in many cases, one drug gen- erally alters the metabolism or transport of a second drug. Most DDIs involve alterna- tions in the metabolic pathways within the CYP system. There are two mechanisms involve for the CYPs; that is, through the process of CYP induction which increases drug clearance that causes a decline or loss of therapeutic effi cacy or when one drug inhibits metabolism of another drug. Drug-CYP induction is typically caused by activation of gene transcription via ligand-a ctivated specifi c receptor which eventually leads to an increase in CYP enzyme expression. The three most commonly involved nuclear hormone receptors are (1) PXR, which up-regulates expression of the CYP3A, CYP2B, and CYP2C, (2) CAR, which also results in enhanced expression of the CYP3A, CYP2B, and CYP2C, and (3) AhR, which results in enhanced expression of the CYP1A viii Preface and 1B enzymes. Nuclear hormone receptor activation assays using stable cell lines are described in C haps. 10 – 13 in the second edition. CYP induction can be evaluated using human hepatocytes as described in Chap. 1 4 in the second edition or Chap. 13 in the fi rst edition. Each system has its own advantages and limitations, and the decision to use a particular approach depends upon the goal of the drug evaluation. Drug-inhibition of CYPs is typically caused by reversible and irreversible inhibition mechanisms. In Chaps. 1 4 and 1 5 in the fi rst edition, high-throughput screening assays for 13 individual CYPs by using fl uorescent substrates and cDNA-expressed enzymes, and 6 individual CYPs using specifi c substrate probes and human liver microsomes were described to measure reversible inhibition mechanisms, respectively. In the second edition, sev- eral assays are described to measure irreversible inhibition mechanisms. C hapters 1 5 and 16 in the second edition outline assays to measure irreversible inhibition (i.e., time- dependent inhibition) using plated and suspendered human hepatocytes while C haps. 17 – 19 use human liver microsomes combined with novel detection methods. A systemic approach is given in Chap. 16 in the fi rst edition to identify mechanism-based CYP inhibitors. Thus, a complete set of assays are available to address many DDI questions that occur due to alterations in the metabolic CYP system pathways. The ATP binding cassette (ABC) superfamily and the solute carrier (SLC) family of transporters play a major role in infl uencing the pharmacokinetics and toxicokinetics of drugs since they are responsible for the effl ux of a plethora of therapeutic drugs, peptides, and endogenous compounds across biological membranes. The ABC subfamily contains nine transporters which have different intracellular localizations, substrate specifi cities, and structures. In Chaps. 20 – 22 in the second edition, in vitro methods for discovering substrates and inhibitors for the P-glycoprotein (P-gp/ABCB1), the breast cancer resistance protein (BCRP), and the multidrug resistance-associated protein 2 (MRP2; ABCC2) are discussed in detail, respectively. The organic anion transporting polypeptides (OATPs) are members of the SLC family of transporters. In Chaps. 2 3 and 2 4 in the second edition, i n vitro assays for discovering substrates and inhibitors of OATP1B1 and OATP1B3, which are predominantly expressed at the sinusoidal membrane of hepatocytes, and OAT1 (SLC22A6), OAT3 (SLC22A8), and OCT2 (SLC22A2), which are primarily expressed in the proximal tubule epithelial cells of the kidney, are discussed in detail, respectively. When these assays are combined with transporter assays from Chaps. 6 and 7 in the fi rst edition, an arsenal of assays are available to understand the major role that transporters play in infl uencing the drug’s pharmacodynamics. In Chaps. 25 – 27 in the second edition, a variety of assays are presented dealing with establishing good in vitro LC/MS/MS assays, LC/MS/MS methods for the identifi ca- tion of metabolites in biological fl uids, and the detection of endogenous and xenobi- otic compounds from biological fl uids using LC/MS/MS and dried blood spot techniques, respectively. The most important clearance pathways for most drugs in humans involve drugs being metabolized by CYP enzymes to more polar compounds that are eventually eliminated in urine. However, CYP enzyme-mediated metabolism can also lead to drug bioactivation resulting in the formation of reactive metabolites that can potentially induce idiosyncratic toxicity by covalently binding to endogenous proteins and nucleic acids before being eliminated from the body. Because reactive metabolites are not stable, direct detection and characterization of them is not technically feasible. Therefore, many assay strategies have been developed to study the bioactivation liability of drug candidates by using trapping reagents that result in the formation of stable adducts which are subsequently characterized Preface ix by tandem mass spectrometry. In Chaps. 2 8 – 30 in the second edition, a variety of assays are presented dealing with drug bioactivation including the utilization of trapping reagents that results in the formation of stable adducts, quantitative methods for detecting reactive metabolites using radioactive and non-radioactive reagents, and screening assays for deter- mining the reactivity of acyl glucuronides. When these assays are combined with reactive metabolites assays from Chaps. 24 and 2 5 in the fi rst edition, a set of assays are available with clear advantages and objectives to help medicinal chemists to optimize lead compounds at an early stage of drug discovery. The failure of NCEs in both clinical development and aftermarket launch for toxicity reasons is still a major concern for pharmaceutical companies. Therefore, toxicity assays that can provide information at an early stage of drug discovery are of major concerns for medicinal chemists to optimize lead compounds. Interaction of drugs with DNAs potentially results in DNA damage or covalent modifi cations which may lead to genotoxicity. In Chaps. 31 and 3 2 in the second edition, a method for detecting DNA damage at the level of individual eukaryotes induced by drugs using the traditional i n vitro Comet assay (neu- tral and alkaline) is presented and a system based in eukaryotic yeast cells that utilize an endogenous DNA damage-responsive gene promoter and a reporter gene fusion to assess the ability of the drugs to damage DNA is presented, respectively. Here the authors provide examples of these assays with detailed procedures used in their laboratory for the analysis and interpretation of assay data. Combining these new versions with DNA damage assays from Chaps. 1 7 – 20 in the fi rst edition, a set of assays are available to medicinal chemists to provide a path forward in early stage drug discovery lead optimization pro- grams. Although the Ames test has long been used to detect mutagens and possible car- cinogens, an improved version of the assay is given in C hap. 3 3 in the second edition as compared to the version in Chap. 21 in the fi rst edition. Also, a new version of the mouse lymphoma assay (MLA) is outlined in C hap. 3 4 in the second edition as compared to the version in Chap. 22 in the fi rst edition. In Chap. 2 3 in the fi rst edition, a high through i n vitro hERG channel assay was presented. Finally, we want to express our tremendous gratitude to all the authors that contributed chapters to this book. Without their time and energy, the second edition of Optimization in Drug Discovery: In Vitro Methods would not have been possible. Spring House, PA, USA G ary W . Caldwell Spring House, PA, USA Zhengyin Yan